It is generally recognized that important technical advances in chemistry, biology and medicine benefit from the ability to perform microanalysis of samples in minute quantities. However, making analytical measurements on minute quantities has long been a challenge due to difficulties encountered with small volume sample handling, isolation of analytes, and micro-analysis of single-cell physiology.
Nanoliter, picoliter, and femtoliter volume studies have been explored in a range of applications involving in vitro and in vivo cellular investigations [R. M. Wightman, et al., Proc. Natl. Acad. Sci. U.S.A. 88:10754(1991); R. H. Chow, et al. Nature 356:60(1992); T. K. Chen, et al. Anal. Chem. 66:3031(1994); S. E. Zerby, et al., Neurochem. 66:651(1996); P. A. Garis, et al. J. Neurosci. 14:6084(1994); G. Chen, et al., J. Neurosci. 15:7747(1995)], electrochemistry [R. A. Clark, et al., Anal. Chem. 69(2):259(1997)], matrix-assisted laser desorption-ionization mass spectrometry [S. Jespersen, et al., Rapid Commun. Mass Spectrom. 8:581(1994)], micro-column liquid chromatography [I. A. Holland, et al., Anal. Chem. 67:3275(1995); M. D. Oates, et al., Anal. Chem. 62:1573(1990)], micro-titration [M. Gratzl, et al Anal. Chem. 65:2085(1993); C. Yi, et al., Anal. Chem. 66:1976(1994)], and capillary electrophoresis [M. Jansson, et al., J. Chromatogr. 626:310(1992); P. Beyer Hietpas, et al. J. Liq. Chromatogr. 18:3557(1995)].
Clark, et al. [Anal. Chem. 69(2):259(1997)] has disclosed a method for fabricating picoliter microvials for electrochemical microanalysis using conventional photolithographic masking and photoresist techniques to transfer mold polystyrene microvials on silicon wafer templates. These microvials typically exhibit non-uniformity in size and shape due to the difficulty in controlling the resist etching of the molding surface and the transfer molding process.
Park, et al. [Science 276:1401(1997)] has disclosed a modified lithographic method for producing arrays of nanometer-sized holes using polystyrene-polybutadiene, ordered, diblock copolymers as masks in reactive ion etching of silicon nitride. This multi-step method is capable of producing arrays of picoliter-sized holes which are typically 20 nanometers in diameter and 20 nanometers deep with a spacing of 40 nanometers. Hole densities of up to 10.sup.11 holes/cm.sup.2 are disclosed. The range of sizes and spacings of the holes produced by this method is limited by the size of the copolymer microdomains. Uniformity of hole size and spacing is difficult to maintain with this method due to difficulties in controlling the etching method employed to form the holes.
Deutsch, et al. [Cytometry 16:214(1994)] have disclosed a porous electroplated nickel microarray comprised of micron-sized conical holes in blackened nickel plate. Hole sizes range from a 7 um upper diameter to a 3 um lower diameter with an 8 um depth. The array is used as a cell carrier for trapping individual cells while studying the responses of individual cells to changes in their microenvironment. In U.S. Pat. No. 4,772,540, Deutsch, et al., have also disclosed a method for making such an array using a combined photoresist and electroplating technique.
Coming Costar Corp. (Acton, Mass.) produces a commercial microwell array for miniaturized assays under the trademark PixWell.TM.. These arrays are made from microformed glass plates and comprise 40 um diameter by 20 um deep tapered wells with a well density of 4356 wells/cm.sup.2.
Microwell arrays have particular utility in the study of living cells. In cell research, the measurement of responses of individual cells to changes or manipulations in their local environment is desirable. Any method or device designed for such studies must provide for the capability of maintaining cell viability, identifying the location of individual cells, and correlating response measurements with individual cells.
Due to the availability of viable fluorescent probes for intracellular studies, fluorescence measurements of living cells have significant utility in the study of cell functions. Thus fluorescence optical measurements are often utilized in cell studies where three generic methods of cell measurement are available, comprising bulk measurements of cell populations, dynamic measurements of cell populations or individual cells, and static measurements of individual cells.
The characteristics of an entire cell population as a whole can be studied with bulk measurements of sample volumes having a plurality of cells. This method is preferred where cell populations are very homogeneous. A generally recognized limitation of this method is the presence of background fluorescence which reduces the sensitivity of measurements and the inability of distinguishing differences or heterogeneity within a cell population.
Flow cytometry methods are often employed to reduce problems with background fluorescence which are encountered in bulk cell population measurements [M. R. Gauci, et al., Cytometry 25:388(1996); R. C. Boltz, et al., Cytometry 17:128(1994)]. In these methods, cell fluorescence emission is measured as cells are transported through an excitation light beam by a laminar flowing fluid. Flow cytometry methods may be combined with static methods for preliminary sorting and depositing of a small number of cells on a substrate for subsequent static cell measurements [U.S. Pat. No. 4,009,435 to Hogg, et al.; Kanz, et al., Cytometry 7:491(1986); Schildkraut, et al., J. Histochem Cytochem 27;289(1979)].
Gauci, et al., disclose a method where cell size, shape and volume is measured by light scattering and fluorescent dyes are utilized to determine protein content and total nucleic acid content of cells. This method fuirther provides for counting and sizing various cells at a rate of approximately 100 cells per second.
Flow cytometry techniques are generally limited to short duration, single measurements of individual cells. Repetitive measurements on the same cell over time are not possible with this method since typical dwell times of a cell in the excitation light beam are typically a few microseconds. In addition, the low cumulative intensity from individual cell fluorescence emissions during such short measurement times reduces the precision and limits the reliability of such measurements.
Regnier, et al., [Trends in Anal. Chem. 14(4):177(1995)] discloses an invasive, electrophoretically mediated, microanalysis method for single cell analysis. The method utilizes a tapered microinjector at the injection end of a capillary electrophoresis column to pierce an individual cell membrane and withdraw a sample of cytoplasm. The method measures cell contents, one cell at a time. The method is generally limited to the detection of easily oxidized species.
Hogan, et al., [Trends in Anal. Chem. 12(1):4(1993)] discloses a microcolumn separation technique which may be utilized in combination with either a conventional gas chromatograph-mass spectrometer, micro thin layer chromatography or high pressure liquid manipulation of small cellular volumes. The sensitivity of the method is limited and may require pre-selection of target compounds for detection.
Static methods are generally the preferred method for measurements on individual cells. Measurement methods range from observing individual cells with a conventional optical microscope to employing laser scanning microscopes with computerized image analysis systems [see L. Hart, et al., Anal. Quant. Cytol. Histol. 12:127(1990)]. Such methods typically require the attachment of individual cells to a substrate prior to actual measurements. Problems are typically encountered in attaching single cells or single layers of cells to substrates and in maintaining cells in a fixed location during analysis or manipulation of the cell microenvironment. Additionally, repetitive measurements on individual cells typically require physically indexing the location of individual cells and providing a mechanism for scanning each cell sequentially and returning to indexed cell locations for repeated analysis of individual cells.
Huang, et al., [Trends in Anal. Chem., 14(4)158(1995)] discloses a static electrochemical method and electrode for monitoring the biochemical environment of single cells. The method requires fabrication and manual positioning of a microelectrode reference and working electrode within the cell. The method has been used to detect insulin, nitric oxide and glucose inside single cells or external to the cells. The method is generally limited to the study of redox reactions within cells.
Ince, et al. [J. Immunol. Methods 128:227(1990)] disclose a closed chamber device for the study of single cells under controlled environments. This method employs a micro-perfusion chamber which is capable of creating extreme environmental conditions for cell studies. Individual cells are held in place by two glass coverslips as various solutions are passed through the chamber. One limitation of the method is the difficulty in eliminating entrapped gas bubbles which cause a high degree of autofluorescence and thus reduces the sensitivity of measurements due to background fluorescence.
In an attempt to overcome the limitations encountered with conventional static methods, Deutsch, et al., [Cytometry 16:214(1994)] and Weinreb and Deutsch, in U.S. Pat. Nos. 4,729,949, 5,272,081, 5,310,674, and 5,506,141, have disclosed an apparatus and method for repetitive optical measurements of individual cells within a cell population where the location of each cell is preserved during manipulation of the cell microenvironment.
A central feature of the apparatus disclosed by Deutsch, et al., is a cell carrier, comprising a two dimensional array of apertures or traps which are conical-shaped in order to trap and hold individual cells by applying suction. The cell carrier is typically fabricated by the combined electroplating-photoresist method disclosed in U.S. Pat. No. 4,772,540 to Deutsch, et al. The purpose of the cell carrier is to provide a means for maintaining the cells in fixed array locations while manipulating the cell environment. Individual cells are urged into cell carrier holes by suction and the wells are subsequently illuminated with a low-intensity beam of polarized light that reads back-emitted polarization and intensity. Measurements are compared when two different reagents are sequentially reacted with the cells. The method as disclosed requires two separate cell carriers for both a baseline control and analyte measurement.
The method and device of Deutsch, et al., have been employed by pathologists in diagnostic tests to determine the health and viability of cell samples taken from patients. The method and device have been applied to both cancer screening [Deutsch, et al., Cytometry 16:214(1994), Cytometry 23:159(1996), and European J. Cancer 32A(10):1758(1996)] and rheumatoid arthritis [Zurgil, et al., Isr. J. Med. Sci. 33:273(1997)] in which fluorescence polarization measurements are used to differentiate lymphocytes of malignant versus healthy cells based on changes in the internal viscosity and structuredness of the cytoplasmic matrix induced by exposure to tumor antigen and mitogens.
The method and device disclosed by Deutsch, et al., requires employment of a scanning table driven by three stepping motors and a computer control system for mapping, indexing and locating individual cells in the cell carrier. The use of such mechanical scanning methods introduces limitations in reproducibility and accuracy of measurements due to conventional mechanical problems encountered with backlash and reproducible positioning of individual cell locations for repeated measurements. In addition, mechanical scanning of the entire array prolongs the measurement time for each cell in the array.
The method disclosed by Deutsch, et al., is further limited by the use of fluorescence polarization measurements which have certain intrinsic limitations due to the significant influence of various optical system components on polarization as the fluorescence emission response is passed from the cell carrier to optical detectors. Birindelli, et al. [European J. Cancer 33(8):1333(1997)], has also identified limitations in this method due to fluctuations in electropolarisation values which require taking averages of at least three measurement scans for each condition so as to obtain reliable measurements. In addition, for cell studies, polarization measurements are generally limited to cell responses which produce sufficient changes in cytoplasm viscosity to produce a detectable change in polarization. Since not all cell responses are accompanied by detectable viscosity changes, the method is further limited to the cell activities which create such viscosity changes in the cytoplasm.
Zare, et al., [Science 267:74(1995); Biophotonics International, March-April, p17 (1995)] discloses a biosensor system based on the response of living cells to complex biological materials fractionated by a microcolumn separation technique. Cells which were positioned on a glass cover slip were treated with a fluorescent probe and subsequently shown to be sensitive to a series of biological compounds including acetylcholine, bradykinin, and adenosine triphosphate as well as changes in intracellular calcium levels.
Yeung, et al. [Acc. Chem. Res. 27:409(1994)] has reviewed a number of methods for single cell response studies and has observed a significant variation and heterogeneity within cell populations based on analyte measurements. For example, the reference discloses a capillary electrophoresis method for exposing cells to biologically reactive compounds, extracting the intracellular fluid of individual cells produced in response to such compounds, and identifying analytes from migration times in the capillary column. Other fluorescence-based assays are also disclosed. Significant cell-to-cell variations and heterogeneity in individual cell responses within a cell population were observed which differences could provide a means for discriminating between biological and chemical compounds in contact with individual cells.
McConnell, et al. [Science, 257:1906(1992)], disclose a microphysiometer device known as the "Cytosensor" which uses a light addressable potentiometer sensor to measure the rate at which cells acidify their environment. This sensor acts as miniaturized pH electrode for monitoring cell responses which produce detectable changes in local pH. The disclosed device is limited to the measurement of proton excretions from cells and thus is only capable of detecting acidic cell responses to analytes.
U.S. Pat. No. 5,177,012 to Kim, et al., disclose a biosensor for the determination of glucose and fructose. The biosensor is produced by treating whole cells with an organic solvent and immobilizing the treated cells residue on a support to form a whole cell membrane which is applied to a pH electrode.
U.S. Pat. No. 5,690,894 to Pinkel, et al., discloses a biosensor which employs biological "binding partners", materials such as nucleic acids, antibodies, proteins, lectins and other materials derived from cells, tissues, natural or genetically-engineered organisms. These agents are used in conjunction with a fiber optic array where each species of binding partners is uniquely addressed by a group of fibers within the fiber optic bundle which is coupled to an optical detector. The array was designed for screening of extensive arrays of biological binding partners.
While many of the prior art methods provide for the analysis of either single cells or populations of cells and some of these methods provide for monitoring cell responses to target analytes, none of the disclosed methods provides for employing large populations of monocultures or mixed populations of living cells for simultaneously monitoring the responses of individual cells to biological stimuli produced by chemical and biological analytes. Thus there is a need for a biosensor array and method which efficiently utilizes the ability of populations of living cells to respond to biologically significant compounds in a unique and detectable manner. Since the selectivity of living cells for such compounds has considerable value and utility in drug screening and analysis of complex biological fluids, a biosensor which makes use of the unique characteristics of living cell populations would offer distinct advantages in high throughput screening of combinatorial libraries where hundreds of thousands of candidate pharmaceutical compounds must be evaluated. In addition, such a sensor would be useful in monitoring bioprocesses and environmental pollution where the enhanced sensitivity of living cells to their environment can be exploited.